The epidermal growth factor receptor (EGFR/ErbB) family of receptor tyrosine kinases includes four family members in humans: EGFR (HER1, ErbB1), HER2 (ErbB2, Neu), HER3 (ErbB3), and HER4 (ErbB4). Each family member consists of an extracellular ligand-binding region, a single membrane-spanning region, a cytoplasmic tyrosine kinase, and a C-terminal tail of ~230 amino acids. All EGFR/ErbB family members are essential for normal embryonic development, and abnormal activity of each ErbB has been associated with human cancer. In particular, overexpressed or active EGFR and HER2 are associated increased severity of lung, colon, and head-and-neck cancers (EGFR) or breast cancer (HER2), and several drugs targeting EGFR and HER2 are FDA-approved anticancer therapies. The canonical model of EGFR activity is that a ligand binding to the extracellular region stabilizes a specific dimeric conformation of the receptor in which its kinase activity is stimulated, which results in phosphorylation of the receptor C-tail and other substrates, changes in the localization and/or activity of downstream effectors, and initiation of signaling cascades that alter cell growth and differentiation. HER2 and HER3 are atypical ErbBs in that HER2 has no known ligand and HER3 has little or no kinase activity owing to key mutations in its kinase domain. Both HER2 and HER3 appear to function as heterodimeric partners of other ErbB family members, however, and the HER2/HER3 heterodimer is the most potent signaling and oncogenic ErbB pairing by many criteria. Much has been learned about EGFR/ErbB family members from structural and functional studies of ErbB fragments, but our knowledge of intra- and intermolecular regulation of EGFR/ErbB activity has suffered from difficulties studying intact ErbBs. In the last five years our laboratories have collaborated to express and purify near milligram amounts of stable, active full-length and near full-length forms of EGFR. We have used this material to provide a rigorous characterization of EGFR enzymatic activity in ligand-activated and inhibited states and have begun to provide a characterization of the effects of EGFR-activating mutations found in lung cancer. We propose to provide a comprehensive, mechanistic characterization of ErbB activity and its regulation in normal and diseases states by extending our enzymological and functional studies of purified EGFR to determine (i) the effects of specific cancer-associated mutations of EGFR on EGFR activity and, through combination of these mutations with additional mutations targeting specific regions of EGFR, the mechanism by which these mutations exert their effects, (ii) the ability of EGFR inhibitors and combinations of inhibitors to target specific forms of activated EGFR, (iii) the influence of EGFR glycosylation and the EGFR C-tail on EGFR activity and substrate specificity, and (iv) the in vitro substrates of EGFR and mutant EGFRs using a 17,000 protein microarray. We have recently extended our expression and purification to HER2, HER3, and HER4, and we propose to carry out similar mechanistic and enzymological studies of the intrinsic and regulated activity of these ErbBs.